Localization of sequence segments forming a kappa-bungarotoxin-binding site on the alpha 3 neuronal nicotinic receptor.
To identify the sequence segments of the alpha 3 subunit of the neuronal nicotinic acetylcholine receptor (N-nAChR) forming the binding site for the cholinergic antagonist kappa-bungarotoxin (kappa-BGT), overlapping peptides corresponding to the complete alpha 3 sequence were tested for their ability to bind 125I-labeled kappa-BGT. Two peptides located within the N-terminal extracellular domain specifically bound kappa-BGT in a solid phase assay, i.e. peptide N alpha(3)51-70 with a Kd approximately 300 nM and peptide N alpha(3)1-18 with slightly lower affinity (Kd approximately 500 nM). Preincubation of 125I-kappa-BGT with peptides N alpha(3)51-70 or N alpha(3)1-18 resulted in greater than 90% inhibition of kappa-125I-BGT binding to native N-nAChR expressed on the neuronal cell line PC12. Under the same conditions, two additional peptides, N alpha(3)180-199 and N alpha(3)183-201, were found to inhibit kappa-125I-BGT binding to PC12 by approximately 50%. These latter peptides represent sequences that are homologous to those shown previously to bind alpha-bungarotoxin. Peptide N alpha(3)51-70 (400 microM) also reduced by approximately 4-fold the observed rate of association of kappa-BGT to PC12 cells. The results of these experiments identify sequence segments of the alpha 3 subunit which are likely to interact with kappa-BGT and may indicate the relative contribution that these segments make in the formation of the high affinity kappa-BGT-binding site of this N-nAChR subtype.
McLane
K E
KE
Department of Biochemistry, College of Biological Sciences, University of Minnesota, Saint Paul 55108.
Tang
F
F
Conti-Tronconi
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010673:Pheochromocytoma; D051381:Rats; D011978:Receptors, Nicotinic / Q000378:metabolism*
D002038:Bungarotoxins; D009842:Oligopeptides; D011978:Receptors, Nicotinic